An important aspect of immune responses at mucosal surfaces is the production of secretory IgA (S-IgA) and its transport across the epithelium. This S-IgA response represents the first line of defense against the invasion by viral and bacterial pathogens. The mucosal immune system is an integrated network of tissues, cells and effector molecules which function to protect the host from those pathogens. Because of the presence of unique interconnected mucosal inductive and effector sites the mucosal immune system is separated from the peripheral immune system. Thus, the induction of peripheral immune responses by parenteral immunization does not result in significant mucosal immunity; however, mucosal immunization is capable of inducing protective immunity in both external secretions and peripheral immune compartments [2, 3].
The primary reason for using a mucosal route of vaccination is that most infections affect or start from a mucosal surface and that in these infections, topical application of a vaccine is often required to induce a protective immune response [1].
During work in the 1920s on the production of animal sera for human therapy, it was discovered that certain substances, notably aluminum salts, added to antigens greatly enhances antibody production—that is it acts as an adjuvant. Activation of innate immune responses is a prerequisite for an adjuvant function and a much needed component in any vaccine. Currently, a very limited spectrum of vaccine adjuvants are used commercially, with aluminum salts still being by far the largest group. With modern understanding of the process leading to triggering and the development of immunologic memory, considerable efforts have been made to produce better adjuvants (Immunology 7′ th edition, 18-19, 2006, Ivan Roitt et al.).
Recently, much effort has been focused on inducing effective immune responses in mucosal tissues: however, most protein antigens (Ags) are rather weak immunogens when given via the mucosal route. In this regard, the coadministration of mucosal adjuvants, such as cholera toxin (CT), has been shown to effectively support Ag-specific mucosal immune responses. Thus, the development of effective and reliable mucosal adjuvants is a focus for a new generation of vaccines [3].
The CT contains 5 GM1 binding (B) subunits, an active (A1) subunit, and a bridging piece (A2) that links A1 to the 5B subunits. After it enters the cell, the A1 subunit enzymatically transfers ADP ribose from NAD to Gs protein, that regulates the adenylate cyclase system which is located on the inside of the plasma membrane of mammalian cells. The A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein forming Gs-ADPR from which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates adenylate cyclase, the enzyme remains continually activated and stimulates adenylate cyclase, which results in the formation of large quantities of intracellular cyclic AMP (cAMP) [4].
The increase in cAMP often acts to immunomodulate many immune reactions such as enhanced antigen presentation by various antigen presenting cells (APCs) [5], promotion of isotype differentiation in B cells leading to increased IgA formation [6], and up-regulation of surface expression of CD80 and CD86 on the APC [7, 8].
CT is a strong systemic and mucosal adjuvant that greatly enhances IgG and IgA immune responses. Since the 1980s, the ability of CT to act as a mucosal adjuvant has been confirmed by a number of investigators with a variety of Ags [9]. The adjuvant properties of CT have been studied in a mouse model using both in vivo and in vitro experimental systems [10-13].                However, CT is a potent enterotoxin. For example, as little as 5 nanograms of purified CT administered orally is sufficient to induce significant diarrhea in humans, suggesting that enterotoxicity may seriously limit the practical use of CT [14].        
Several studies were done to separate enterotoxicity from adjuvanticity. Mutants of CT have been constructed in attempts to dissociate the enterotoxic effects of these molecules from their adjuvant activity. Mutations in both the active site and the protease site of these two molecules have been examined and a number of different mutants of CT have been characterized [15-17].
A mutant CT (mCT; S61F and E112K) failed to induce adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated ileal loops, and were thus nontoxic [18]. The mCT S61F also acts as a mucosal adjuvant by inducing CD41. Th2 cells secrete IL-4, IL-5, IL-6 and IL-10, which provide effective help for Ag-specific mucosal S-IgA, as well as serum IgG1, IgE and IgA Ab responses. Mucosal adjuvant activity of mCT S61F and E112K has been demonstrated using several Ags including ovalbumin, tetanus toxoid and influenza virus [18].
A fusion protein that combines the enzymatically active CTA1 with the Ig binding D region (DD) of staphylococcal protein A (CTA1-DD) was shown to augment specific serum as well as mucosal Ab responses to soluble protein Ags. CTA1-DD retains full enzymatic activity of the CTA1-subunit in a B cell-targeted fusion protein that lacks CTB and, thus, cannot bind to the GM1-ganglioside receptor [7, 19].
CTA1-DD was found to be completely non-toxic and, despite its more selective binding properties, had mucosal and systemic adjuvant effects comparable to that of intact CT (U.S. Pat. No. 5,917,026). However, the latter construct exhibits a selective cell binding moiety, DD (Staphylococcus aureus protein A binding site), aimed at directing the CTA1 moiety to cells expressing a receptor for DD, and specifically to subpopulations of B lymphocytes and dendritic cells. CTA1-DD was also shown to be highly immunogenic to CTA1 moiety and this enhancement of CTA1-specific serum Abs was essential to act as an immunoenhancing adjuvant (20)
Thus, there is a continuing need for more effective and reliable mucosal adjuvants for use in vaccines to produce mucosal and systemic immune effects.